Comparison of predictive methods and biological validation for qPCR reference genes in sunflower leaf senescence4 transcript analysis.

FERNANDEZ P; DI RIENZO J; MOSCHEN S; DOSIO, G.A.A; AGUIRREZÁBAL L.A.N; HOPP, HE; PANIEGO, N; HEINZ, R

PLANT CELL REPORTS

SPRINGER

Año: 2011 vol. 30 p. 63 - 74

0721-7714

The selection and validation of reference genes 10 constitute a key point for gene expression analysis based onconstitute a key point for gene expression analysis based on 11 qPCR, requiring efficient normalization approaches. In thisqPCR, requiring efficient normalization approaches. In this 12 work, the expression profiles of eight genes were evaluatedwork, the expression profiles of eight genes were evaluated 13 to identify novel reference genes for transcriptional studiesto identify novel reference genes for transcriptional studies 14 associated to the senescence process in sunflower. Threeassociated to the senescence process in sunflower. Three 15 alternative strategies were applied for the evaluation ofalternative strategies were applied for the evaluation of 16 gene expression stability in leaves of different ages andgene expression stability in leaves of different ages and 17 exposed to different treatments affecting the senescenceexposed to different treatments affecting the senescence process: algorithms implemented in geNorm, BestKeeper 1818 software, and the fitting of a statistical linear mixed model. 1919 The results show that geNorm suggested the use of all 2020 combined genes, although identifying a-TUB1 as the most 21a-TUB1 as the most 21 stable expressing gene. BestKeeper revealed a-TUB and 22a-TUB and 22 b-TUB as stable genes, scoring b-TUB as the most stable 23-TUB as stable genes, scoring b-TUB as the most stable 23 one. The statistical linear mixed model identified a-TUB, 24a-TUB, 24 actin, PEP, and EF-1a as stable genes in this order. The 25, PEP, and EF-1a as stable genes in this order. The 25 model-based approximation allows not only the estimation 2626 of systematic changes in gene expression, but also the 2727 identification of sources of random variation through 2828 the estimation of variance components, considering the 2929 experimental design applied. Validation of a-TUB and 30a-TUB and 30 EF-1a as reference genes for expression studies of three 31-1a as reference genes for expression studies of three 31 sunflower senescence associated genes showed that the first 3232 one was more stable for the assayed conditions. We con- 3333 clude that, when biological replicates are available, 3434 LMModel allows a more reliable selection under the 3535 assayed conditions. This study represents the first analysis 3636 of identification and validation of genuine reference genes 3737 for use as internal control in qPCR expression studies in 3838 sunflower, experimentally validated throughout six differ- 3939 ent controlled leaf senescence conditions.